During embryonic development, neurons must project their axons along specific paths to make precise connections with their synaptic targets. A key challenge is to identify the involved factors and to understand their signaling mechanisms. The navigation of axonal tips ("growth cones"), both in vitro and in vivo, is guided by insoluble factors in the surrounding extracellular matrix (ECM) or cells and by concentration gradients of diffusing factors. Several families of guidance molecules have been identified and purified, such as netrins, semaphorins, ephrins and Slit proteins. The elucidation of pathfinding decisions in vivo is complicated by the simultaneous presence of different guidance factors in a dynamic environment. Recent studies suggest that the combined effects of these signals are often not obvious. For example, the growth cone attraction to netrin-1 can be converted to repulsion by laminin-1, a contact-attractant. The attractive effects of netrin-1 can also be silenced by the interaction between the cytoplasmic domains of netrin-1 receptor and Slit2 receptor while Slit2 alone did not induce a direct response. In addition, the interaction of netrin-1 and Slit2 pathways is developmentally regulated: older neurons become repelled by Slit2 or by a combination of netrin-1 and Slit2 but show no response to netrin-1 alone. Due to experimental complexity, most studies have only been able to investigate one or two signaling molecules at a time. A traditional in vitro assay records the growth cone responses to gradients of soluble factors delivered by a micro-positioned pipette. This assay provides a means to study single cells, but it generates gradients that are imprecise and evolve in time, and it yields results at very low throughput (-1 cell/hour). Here we propose to build a microfluidic device that will allow us to study multiple axon guidance factors simultaneously on cultured neurons. We will: a) design a combinatorial diffusive mixer that will generate multiple combinations of gradients of different axon guidance molecules, b) record the responses of neurons to each combination using time-lapse imaging, and c) employ automated analysis tools to process large amounts of images. The microfluidic device will allow us to: 1) generate stable concentration gradients, 2) precisely control spatiotemporal changes of the gradients, and 3) simultaneously study many different combinations of gradients at high throughput. Consequently this study will probe axon guidance factor interactions of unprecedented complexity and will significantly further our understanding of neural development. It may also shed light on new clinical therapies for certain neurological disorders and for nerve regeneration after injury.